The primary objective of this proposal is to study squamous-cell carcinomas of the vulva, vagina, cervix and anus to provide a unified picture of the pathogenesis of human papillomavirus (HPV) infection of the anogenital epithelium. The specific aims of this proposal are, in summary: 1) To investigate the pathobiology of genital HPV infection in women by relating the state of the virus genome and expression of virus E6/E7 genes to histopathologic changes seen throughout vulvar and vaginal cancer excision specimens and cervical conization biopsies, 2) To study the pathogenesis of HPV-associated anal cancers, in men and women, both by mapping the presence of HPV DNA and by defining the topography of E6/E7 expression in squamous- cell carcinomas of the anal canal and skin, 3) To study the pathobiology of concurrent infection with multiple HPV types in a single squamous lesion of the anogenital epithelium. This study will use existing formalin-fixed, paraffin-embedded tissue specimens for in situ hybridization (ISH) and polymerase chain reaction (PCR) analyses. HPV DNA genomes will be localized in tissue sections by ISH and PCR, and expression of virus E6/E7 genes will be visualized by ISH with exon-specific RNA probes. Small segments of anogenital cancer excision specimens will be separately analyzed by ISH and PCR to precisely define the relationship between virus functions (presence of viral DNA and level of E6/E7 expression) and morphologic changes (degree of intraepithelial neoplasia, microinvasive or invasive cancer) throughout the entire lesion. Histologically normal epithelium from the margins of tumor excisions will be examined by ISH and PCR to determine if diffuse HPV infection exists beyond an area of focal disease. Intraepithelial and invasive squamous-cell lesions of the female anogenital epithelium that contain DNA of more than one HPV type will be studied to define the pathobiology of concurrent infection with multiple viruses. ISH with non-radioactive DNA probes and with radioactive single- stranded RNA probes will be used to localize the different HPV types in a single lesion and to determine if both viruses are transcriptionally active.